Doxorubicin (DOX) is a key chemotherapeutic drug for cancer treatment. The antitumor mechanism of DOX is its action as a topoisomerase II poison by preventing DNA replication. Our study shows that DOX can be involved in epigenetic regulation of gene transcription through downregulation of DNA methyltransferase 1 (DNMT1) then reactivation of DNA methylation-silenced tumor suppressor genes in glioblastoma (GBM). Recent evidence demonstrated that microRNA (miR or miRNA) can mediate expression of genes through post-transcriptional regulation and modulate sensitivity to anticancer drugs. As one of the first miRNAs detected in the human genome, miR-21 has been validated to be overexpressed in GBM. Combination treatment of a chemotherapeutic and miRNA showed synergistically increased anticancer activities which has been proven to be an effective strategy for tumor therapy. In our study, co-treatment of DOX and miR-21 inhibitor (miR-21i) resulted in remarkably increased expression of tumor suppressor genes compared with DOX or the miR-21i treatment alone. Moreover, we demonstrate that combining DOX and miR-21i significantly reduced tumor cell proliferation, invasion and migration in vitro. Our study concludes that combining DOX and miR-21i is a new strategy for the therapy of GBM.